Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit

Na,K-ATPase in right side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the alpha-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents, In the presence of Mg2+ and P-i, Cys(964) was the most accessible for both membrane-impermeable 4-acetamido-4'-maleimidylstilbene-2,2'disulfonic acid (or stilbene disulfonate maleimide, SDSM) and membrane-permeable 7-diethylamino-3-(4'-maleimidyl)-4-methylcoumarin (CPM). In the presence of K+, Cys(964) was modified only by hydrophobic CPM, indicating that the environment around Cys(964) was dif ferent in these two conformations, Cys(964) seems to mark the extracellular border of transmembrane segment Me, Cys(911) in transmembrane segment M8 showed similar behavior; however, it was not so readily modified. Complete modification of Cys(964) and Cys(911) causes only partial (about 50%) inactivation of both ATPase activity and Rb+ (or K+) occlusion, indicating that the effect on cation occlusion is indirect and not within the occlusion cavity, The ATP binding capacity remains unaltered by the modifications. Treatment of the K+-stabilized post-tryptic preparation of purified Na,K-ATPase revealed labeling of several cysteines by CPM, none of which were labeled with SDSM, Removal of K+ ions from the preparation, which we have previously shown is accompanied by release of the M5M6 hairpin to the supernatant (1), causes changes in the organization of the C-terminal 21-kDa fragment, In particular Cys(983) in M10 became labeled by both CPM and SDSM, pointing to a tight association between the C terminus and the M5M6 hairpin of the alpha-subunit.